Inhibition of PIKfyve by YM-201636 Dysregulates Autophagy and Leads to Apoptosis-Independent Neuronal Cell Death

The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P-2), synthesised by PIKfyve, regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex, leading to even a mild reduction in PtdIns(3,5)P-2 results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity, using YM-201636, significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly, YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II, an effect that was potentiated by inhibition of lysosomal proteases, suggesting that alterations in autophagy could be a contributing factor to neuronal cell death

A Novel Labeling Approach Identifies Three Stability Levels of Acetylcholine Receptors in the Mouse Neuromuscular Junction In Vivo

The turnover of acetylcholine receptors at the neuromuscular junction is regulated in an activity-dependent manner. Upon denervation and under various other pathological conditions, receptor half-life is decreased., in our setup the same animals are used throughout the whole measurement period, thereby permitting a dramatic reduction of animal numbers at increased data quality. We identified three stability levels of acetylcholine receptors depending on the presence or absence of innervation: one pool of receptors with a long half-life of ∼13 days, a second with an intermediate half-life of ∼8 days, and a third with a short half-life of ∼1 day. Data were highly reproducible from animal to animal and followed simple exponential terms. The principal outcomes of these measurements were reproduced by an optical pulse-labeling assay introduced recently.A novel assay to determine kinetics of acetylcholine receptor turnover with small animal numbers is presented. Our data show that nerve activity acts on muscle acetylcholine receptor stability by at least two different means, one shifting receptor lifetime from short to intermediate and another, which further increases receptor stability to a long lifetime. We hypothesize on possible molecular mechanisms

Functional Interchangeability of Late Domains, Late Domain Cofactors and Ubiquitin in Viral Budding

The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. METHODOLOGY: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. PRINCIPAL FINDINGS: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. CONCLUSIONS: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively

Clathrin Facilitates the Morphogenesis of Retrovirus Particles

The morphogenesis of retroviral particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for particle assembly and maturation. In addition, a number of cellular proteins are found in retrovirus particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of particle assembly and reduction in virion infectivity. For each retrovirus examined, clathrin incorporation appeared to be important for optimal replication. These data indicate that a number of retroviruses employ clathrin to facilitate the accurate morphogenesis of infectious particles. We propose a model in which clathrin contributes to the spatial organization of Gag and Pol proteins, and thereby regulates proteolytic processing of virion components during particle assembly

Exploring the role of competing demands and routines during the implementation of a self-management tool for type 2 diabetes: A theory-based qualitative interview study

Background The implementation of new medical interventions into routine care involves healthcare professionals adopting new clinical behaviours and changing existing ones. Whilst theory-based approaches can help understand healthcare professionals’ behaviours, such approaches often focus on a single behaviour and conceptualise its performance in terms of an underlying reflective process. Such approaches fail to consider the impact of non-reflective influences (e.g. habit and automaticity) and how the myriad of competing demands for their time may influence uptake. The current study aimed to apply a dual process theoretical approach to account for reflective and automatic determinants of healthcare professional behaviour while integrating a multiple behaviour approach to understanding the implementation and use of a new self-management tool by healthcare professionals in the context of diabetes care. Methods Following Diabetes UK’s national release of the ‘Information Prescription’ (DUK IP; a self-management tool targeting the management of cholesterol, blood pressure and HbA1c) in January 2015, we conducted semi-structured interviews with 13 healthcare professionals (general practitioners and nurses) who had started to use the DUK IP during consultations to provide self-management advice to people with type 2 diabetes. A theory-based topic guide included pre-specified constructs from a previously developed logic model. We elicited healthcare professionals’ views on reflective processes (outcome expectations, self-efficacy, intention, action and coping planning), automatic processes (habit), and multiple behaviour processes (goal priority, goal conflict and goal facilitation). All interviews were audio recorded and transcribed verbatim and all transcripts were independently double coded and analysed using content analysis. Results The majority of healthcare professionals interviewed reported strong intentions to use the DUK IP and having formed a habit of using them after a minimum of one month continuous use. Pop-up cues in the electronic patient records were perceived to facilitate the use of the tool. Factors that conflicted with the use of the DUK IP included existing pathways of providing self-management advice. Conclusion Data suggests that constructs from dual process and multiple behaviour approaches are useful to provide supplemental understanding of the implementation of new self-management tools such as the DUK IP and may help to advance behavioural approaches to implementation science

Ca2+ Permeable AMPA Receptor Induced Long-Term Potentiation Requires PI3/MAP Kinases but Not Ca/CaM-Dependent Kinase II

Ca2+ influx via GluR2-lacking Ca2+-permeable AMPA glutamate receptors (CP-AMPARs) can trigger changes in synaptic efficacy in both interneurons and principle neurons, but the underlying mechanisms remain unknown. We took advantage of genetically altered mice with no or reduced GluR2, thus allowing the expression of synaptic CP-AMPARs, to investigate the molecular signaling process during CP-AMPAR-induced synaptic plasticity at CA1 synapses in the hippocampus. Utilizing electrophysiological techniques, we demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460, and found to be dependent on postsynaptic Ca2+ ions through calcium chelator (BAPTA) studies. Surprisingly, Ca/CaM-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDA-receptor dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDA-receptor dependent LTP in control studies. In contrast, inhibitors for PI3-kinase (LY294002 and wortmannin) or the MAPK cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Similarly, postsynaptic infusion of tetanus toxin (TeTx) light chain, an inhibitor of exocytosis, also had a significant inhibitory effect on this form of LTP. These results suggest that distinct synaptic signaling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain

Molecular Constraints on Synaptic Tagging and Maintenance of Long-Term Potentiation: A Predictive Model

Protein synthesis-dependent, late long-term potentiation (LTP) and depression (LTD) at glutamatergic hippocampal synapses are well characterized examples of long-term synaptic plasticity. Persistent increased activity of the enzyme protein kinase M (PKM) is thought essential for maintaining LTP. Additional spatial and temporal features that govern LTP and LTD induction are embodied in the synaptic tagging and capture (STC) and cross capture hypotheses. Only synapses that have been "tagged" by an stimulus sufficient for LTP and learning can "capture" PKM. A model was developed to simulate the dynamics of key molecules required for LTP and LTD. The model concisely represents relationships between tagging, capture, LTD, and LTP maintenance. The model successfully simulated LTP maintained by persistent synaptic PKM, STC, LTD, and cross capture, and makes testable predictions concerning the dynamics of PKM. The maintenance of LTP, and consequently of at least some forms of long-term memory, is predicted to require continual positive feedback in which PKM enhances its own synthesis only at potentiated synapses. This feedback underlies bistability in the activity of PKM. Second, cross capture requires the induction of LTD to induce dendritic PKM synthesis, although this may require tagging of a nearby synapse for LTP. The model also simulates the effects of PKM inhibition, and makes additional predictions for the dynamics of CaM kinases. Experiments testing the above predictions would significantly advance the understanding of memory maintenance.Comment: v3. Minor text edits to reflect published versio

Meta-Analysis of the Reasoned Action Approach (RAA) to Understanding Health Behaviors

YesBackground: Reasoned action approach (RAA) includes subcomponents of attitude (experiential/instrumental), perceived norm (injunctive/descriptive), and perceived behavioral control (capacity/autonomy) to predict intention and behavior. Purpose: To provide a meta-analysis of the RAA for health behaviors focusing on comparing the pairs of RAA subcomponents and differences between health protection and health-risk behaviors. Methods: The present research reports a meta-analysis of correlational tests of RAA subcomponents, examination of moderators, and combined effects of subcomponents on intention and behavior. Regressions were used to predict intention and behavior based on data from studies measuring all variables. Results: Capacity and experiential attitude had large, and other constructs had small-medium-sized correlations with intention; all constructs except autonomy were significant independent predictors of intention in regressions. Intention, capacity, and experiential attitude had medium-large, and other constructs had small-medium-sized correlations with behavior; intention, capacity, experiential attitude, and descriptive norm were significant independent predictors of behavior in regressions. Conclusions: The RAA subcomponents have utility in predicting and understanding health behaviors